This application claims the benefit of Korean Patent Application No. 10-2004-0001963, filed on Jan. 12, 2004, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
1. Field of the Invention
The present invention relates to a method for immobilizing a biomolecule on a microarray substrate and a biomolecule microarray produced using the method.
2. Description of the Related Art
The term “microarray” refers to a substrate having specific molecules immobilized on predetermined regions at a high density. Examples of the microarray include, for example, a polynucleotide microarray and a protein microarray. Such microarrays are well known in the art, and examples are disclosed in U.S. Pat. Nos. 5,445,934 and 5,744,305. Generally, microarrays are produced by using photolithographic technology. In the photolithographic method, a predetermined region of a substrate coated with a monomer having a removable protective group is exposed to an energy source to remove the protective group from the monomer, and then a second monomer having a removable protective group is coupled to the monomer. The process of exposure to an energy source, removal of the protective group, and coupling of a monomer is repeated to produce a desired polynucleotide on the substrate. Alternatively, microarrays are produced using a method in which a previously synthesized polynucleotide is immobilized at a predetermined location on a substrate, such as a spotting method, a piezoelectric printing method, for example, using an inkjet printer, and a micro-pipetting method, etc. This approach permits biomolecules to be freely arranged, and thus is widely used.
In general, it is difficult to immobilize biomolecules on a surface of a substrate, such as glass or plastics. In conventional methods, in order to immobilize a previously synthesized biomolecule to a solid substrate, the substrate surface was treated to have specific functional groups. Examples of the functional groups include an amino group, an aldehyde group, an epoxide group, and an ester group.
Methods including coating a solid substrate with functional groups and then immobilizing activated biomolecules thereon have been proposed. For example, U.S. Pat. No. 5,350,800 discloses a method including activating a carboxyl group of a carboxyl group containing protein, for example, heparin or laminin using carbodiimide and reacting the resultant product with a solid substrate coated with an amine group to immobilize the product. U.S. Pat. No. 5,760,130 discloses a method including immobilizing a polynucleotide activated with phosphorimidazolide on a glass substrate coated with an amino group. However, in the conventional methods, since the substrate coated with an amino group is used, activation of the biomolecule is required.
U.S. Pat. No. 5,760,130 discloses a method for immobilizing a polynucleotide on a plastic plate which has a carboxyl group. In this method, for example, “Sumilon” MS-3796F and MS-3696F (available from Sumitomo Bakeliet), which have both a carboxyl group and an amino group, can be used. However, in this method, since the plastic plate has a carboxyl group, there is a problem that a density of carboxyl group is too low. Further, this method disclosed in U.S. Pat. No. 5,760,130 is intended to synthesize cDNAs using a support having polynucleotides immobilized thereon. Thus, it is not required to measure fluorescence intensity for analysis of a target biomolecule as in a microarray. The patent does not list a need for introducing the carboxyl groups at a high density. Thus, the present inventors conducted vigorous research to increase fluorescent signal intensity obtained from an analysis of a target biomolecule by immobilizing the biomolecule on a microarray substrate at a high density, and discovered a method for immobilizing a biomolecule on a substrate at a high density by using a carboxylic anhydride.